Abstract

In order to evaluate the potential toxicity of dispersant application in an oil slick in near-shore areas, this study presents an experimental system designed to perform toxicity tests on fish. Three possible oil spill scenario issues during an oil spill were tested on juvenile sea bass (Dicentrarchus labrax); the Water Soluble Fraction of oil; the Mechanical Dispersion of oil; the Chemical Dispersion of oil. Preliminary toxicity assays suggest that the experimental system is appropriate to assess the three experimental conditions during a period of 24 hours. This experimental system allowed obtaining “quick” and relevant results of acute toxicity in an emergency context such an oil spill.

Keywords

Highlights

- A system designed to perform toxicity tests on small water
organisms

- A system devised to simulate the behaviour and the toxicity of the
petroleum following dispersant use.

Introduction

The application of chemical dispersants is a commonly used
technical response in case of oil spill at sea [1]. Dispersant shift the oil
slick from the surface to the water column. In offshore areas, dispersants
are often used since their application shows many environmental
advantages: they increase the natural dilution of oil and consequently the
biodegradation; they also decrease the amount of oil slick grounded on
the shore [2]. However, in near-shore areas, dispersant application is a
controversial countermeasure: the low dilution potential of the oil slick
(in a limited water column depth) is able to enhance the toxicity and
consequently reduces the environmental advantages of dispersant use.
For this reason, in an emergency context such an oil spill, it is necessary to
have as soon as possible toxicity data to evaluate the potential biological
impact. Many studies have evaluated the acute toxicity of dispersant
alone [3-6] or dispersant enhanced water accommodated fractions [7-
10]. These methods presented a major disadvantage. Indeed, they do
not take into account the presence of oil droplets in the water column,
especially in near-shore areas where the mechanical agitation, e.g. wave
action, promotes their formation. Moreover oil droplets are suggested
as a determinant of toxicity [11]. On this basis, an experimental system
adapted from Blackman et al. [12] was proposed to measure the total
petroleum hydrocarbons transferred in the water column and the toxicity
following dispersant application. The present methodological paper
is a prolongation of a preliminary work of Milinkovitch et al. [13] and
discusses the validation of this experimental system to recreate the three
possible oil exposure issues for water column organisms during an oil
spill: (I) the Water Soluble Fraction of oil; (II) the Mechanical Dispersion
of oil; (III) the Chemical Dispersion of oil.

The petroleum used in the study was a crude Arabian light (CAL).
The CAL is oil used in other eco-toxicological studies [14-17]. This oil is
composed of 54% saturated hydrocarbons, 10% polar compounds and
36% aromatic hydrocarbons. To recreate the most realistic conditions
of an oil slick drifting at sea for a few days, the oil was evaporated in a
1 m3 tank for 24 hours. This weathering caused initial evaporation of
the lighter compounds inducing change in the oil composition. The
weathered CAL contained 54% saturated hydrocarbons, 12% polar
compounds and 34% aromatic hydrocarbons. The total evaporation
of oil was approximately 7%. Details of CAL are presented in
supplementary data. The viscosity of the oil was under 5000 cst,
allowing the application of dispersants [18]. Two formulations of thirdgeneration dispersant (1 and 2), manufactured by Total Fluides and
Innospec (Gamlen) were selected.

Experimental system

The experimental system was adapted from Blackman et al.
[12]. It is composed of twelve experimental tanks (units). Each tank
is a 20 L cylinder fitted with a removable central column 77 mm in
diameter that houses a stainless steel shaft and 3 bladed propellers. The
central cylinder has two sets of two apertures situated at the top and
the bottom. The apertures are covered with a metallic mesh screen to
exclude test animals from the propeller housing. The propeller rotates
(at 1000 rpm) to produce a small vortex within the central cylinder,
thus drawing the exposure solutions in through the upper apertures
and expelling them through the lower ones. This homogenization
allowed maintaining oil droplets throughout the water column. The
system is a static water system, e.g. without water supply, maintained
in a temperature controlled room (19°C) and equipped of an aeration
supply. The experimental device complies with the French AFNOR
standard [19] to determine the acute toxicity of a substance.The experimental system was adapted from Blackman et al.
[12]. It is composed of twelve experimental tanks (units). Each tank
is a 20 L cylinder fitted with a removable central column 77 mm in
diameter that houses a stainless steel shaft and 3 bladed propellers. The
central cylinder has two sets of two apertures situated at the top and
the bottom. The apertures are covered with a metallic mesh screen to
exclude test animals from the propeller housing. The propeller rotates
(at 1000 rpm) to produce a small vortex within the central cylinder,
thus drawing the exposure solutions in through the upper apertures
and expelling them through the lower ones. This homogenization
allowed maintaining oil droplets throughout the water column. The
system is a static water system, e.g. without water supply, maintained
in a temperature controlled room (19°C) and equipped of an aeration
supply. The experimental device complies with the French AFNOR
standard [19] to determine the acute toxicity of a substance.

Exposure condition

All the exposure conditions were prepared in 22 L glass exposure
tanks. The four basic exposure conditions to be used were prepared
separately; they included Water Soluble Fraction (WSF), Mechanically
Dispersed oil (MD) and Chemically Dispersed oil using the two
dispersant formulations (CD1 and CD2). The WSF was prepared with
95 g of weathered CAL in 20 L of seawater following the lower energy
method of Singer et al. [20]. Only the liquid phase was used as the
exposure environment. Mechanically dispersed oil (MD) was prepared
using 20 L of sea water and 95 g of weathered CAL; the mixture was
agitated using a propeller mixer (RW 16 Basic IKA) fitted with the same
3 bladed-propeller and using the same rotor speed (1000 rpm) as used
in the experimental system (described in 2.3). Chemically dispersed oil
solutions, using dispersants 1 and 2 (CD1 and CD2), were prepared
using 20 L of sea water, 95 g of weathered CAL, and 5 g of dispersants
1 or 2 respectively (following the manufacturer’s recommended
application ratio of 20:1), and with the same mixing procedure as for
the mechanically dispersed oil solution. Once the exposure media had
been prepared, they were diluted in sea water at six concentrations (0%,
2.4%, 12%, 18%, 24% and 40%) and distributed in the experimental
system.

Experimental design

Groups of 10 fish were exposed to one dilution of each
exposure condition medium for 24 hours in an experimental
tank. Physicochemical parameters (pH, dissolved oxygen, water
temperature, salinity) were monitored. Two exposure conditions
were tested simultaneously: chronologically CD1 and WSF, then CD2
and MD. At the end of the 24 hours exposure period, the fish in each
tank were gently transferred to clean sea water for a 24 hour period,
as recommended by Blackman et al. [12]. For this purpose, 22 L glass
flow-through tanks were used. After 24 hours, each tank was inspected
and dead fish were counted. Fish were considered dead when no gill
movement and no response to a caudal pinch were observed.

Analytical methods

Measurements of total petroleum hydrocarbon (TPH)
seawaterconcentrations: The total petroleum hydrocarbon (TPH)
concentration in each dilution of each exposure medium was assessed
at the beginning (T0) and at the end of the exposure period (T1),using the mean of three replicated measurements for each time point.
TPH concentrations were quantified by spectrophotometry (UV-Vis
spectrophotometer, Unicam at 390 nm) of dichloromethane extracted
samples, as described by Fusey and Oudot [21].

Measurement of the droplet size of dispersed oil: The oil
droplet size distribution (diameter in microns) of CD1, CD2 and MD
conditions were analyzed 6 hours after the beginning of fish exposure
at a nominal concentration of 1250 mg/L. The measurements were
performed by laser granulometry (Malvern Mastersizer 2000) based
on the principle of Fraunhofer according to the intensity of diffracted
radiation, whereby the diffraction angle dependent on the particle size.
A water sample flow rate of 1200 mL/min and an obscuration of 10%
were the conditions used during the measurements.

Statistical analysis

The LC50 values (the TPH concentration of the exposure media that
caused the death of 50% of a group of test animals) were calculated
using the trimmed Spearman-Karber method and a US EPA probit
program, and expressed as values (lower 95% confidence interval
- upper 95% confidence interval). The difference between MD, CD1
and CD2, concerning TPH concentration, was evaluated following
the Quade test procedure: exposure media (MD, CD1 and CD2) were
considered as treatment and the % of stock solutions (0%, 2.4%, 12%,
18%, 24%, 40%) were considered as blocks. Thus, the values obtained
for each exposure environment at several dilutions were considered as
repeated measurements. The statistical analysis was carried out using
Systat 12 software and the significance of the results was ascertained
at α=0.05.

Results and Discussion

The goal of this study was to simulate a possible scenario of
dispersion of a drafting oil spill. For fish exposed to 0% of stock solution,
no mortality was found. Moreover all physicochemical parameters,
temperature (19.1 ± 0.2°C), pH (8.04 ± 0.03), dissolved oxygen (97.5 ±
0.9% of O2 satuation) and salinity (35.2 ± 0.0 PSU), remained constant
throughout the experimental periodfor all exposure conditions. The
possibility to maintain viable juvenile of Dicentrarchus labrax suggests
that the experimental system makes possible to use early life stage in oil
and dispersant toxicity assessment.

When measurements of total petroleum hydrocarbon
concentration for mechanical (MD) and chemical (CD1 and CD2)
dispersion are compared, it appears that the dispersant application
increases significantly the concentration of TPH (mean over 24 hours)
in the water column (Table 1). This result is in accordance with previous
observations obtained in field operations and in situ experimentation
[22,23]. Taken together and very logically, these results show that the
transfer of petroleum from the surface to the water column is increased
when dispersant is applied.

MD

CD1

CD2

% of stock solution

[TPH] (mg/L)

Fish mortality (%)

[TPH] (mg/L)

Fish mortality (%)

[TPH] (mg/L)

Fish mortality (%)

0

nd.

0

nd.

0

nd.

0

2.4

45 (65-25)

0

107 (118-96)

0

102 (130-74)

0

12

214 (235-196)

0

554 (659-449)

0

585 (647-523)

0

18

213 (293-133)

0

971 (1037-905)

50

744 (881-607)

0

24

306 (405-207)

0

1116 (1269-963)

100

964 (1050-878)

30

40

373 (443-302)

0

1547 (1542-1553)

100

1879 (1948-1810)

100

LC50 (mg/L)

n.c.

873 (782-976)

1227 (1091-1379)

The results are expressed as mean concentrations over 24 hours (concentration at T0 - concentration at T1). Respecting Quade test procedures, values obtained for each exposure condition at several % of stock solution are considered as repeated measure and *indicates significant differences (P < 0.05) of TPH concentrations between exposure conditions. LC50 values are expressed as values (lower 95% CI-upper 95% CI). n.d. = not detected. n.c. = not calculable.

Table 1: Fish mortality (%). % of stock solution and total petroleum hydrocarbon concentration (mg/L) in sea water for mechanical dispersion (MD) and chemical dispersion
(CD1 and CD2) during the 24 hour exposures.

Analysis of oil droplet size distribution showed a significant
difference between the two chemically dispersed conditions and the
mechanically dispersed condition (Table 2). This result can explain
a part of the higher TPH concentration of CD1 and CD2 conditions
compared to those observed in MD condition. Indeed, application
of surfactant increases the bioavailability of oil [24]. The two
commercial formulations of dispersant used in this study had different
surfactant concentrations. This difference could conduct to difference
in oil bioavailability for the two chemical dispersion conditions Consequently, oil bioavailability could be one of the reasons of differences observed between MD, CD1 and CD2 and CD2 conditions
in their fish mortalities.

d (0.1)

d (0.5)

d (0.9)

CD1

2.0 ± 0.0 a

5.2 ± 0.0 a

12.5 ± 0.0 a

CD2

1.8 ± 0.0 a

4.1 ± 0.0 a

11.8 ± 0.2 a

MD

107.6 ± 0.9 b

227.9 ± 3.3 b

437.3 ± 11.4 b

In this table, d (0.5), d (0.1) and d (0.9) correspond respectively to the median and the two deciles of a Normal distribution. (n = 7). Differences in letters indicate statistical differences between groups (p < 0.05).

Without dispersant application a part of the oil slick could
solubilised in the water column. The water soluble fraction (WSF) of
oil is commonly used in dispersed crude oil toxicity tests. Thus, we
exposed fish to this treatment in order to compare our results with
those obtained in the literature. Preliminary results of this study
suggest that chemically dispersed oil was more toxic than WSF of oil
since no mortality was found for WSF whatever the percentage of stock
solution tested (0, 2.4, 12, 18, 24 and 40% of stock solution in seawater
for WSF). In our results, no TPH were detected for each % of stock
solution tested. These results are in accordance with other studies using
different experimental approaches [8,10,25- 27]. It can therefore, be
concluded that our experimental system is suitable for assessing the
toxicity of dispersant application.

A rapid decrease in TPH concentration is commonly observed in
offshore field operations [2]. At the opposite, since near-shore areas
have a lower dilution potential and important natural mixing processes
(e.g. wave action), natural dispersion of the oil slick can be maintained
and the oil slick can even be displaced from the surface to the water
column (as described by Lunel [28] during the Braer oil spill). For
example, during Sea Empress and Braer oil spills, high concentration
of oil was observed over more than one week (respectively [29] and
[28]. In this study, the experimental system was under static condition
(e.g. without dilution due to water supply), consequently the evolution
of TPH concentration depends on the turbulent mixing energy. This
allows reproducing different scenarios making possible to simulate a
natural dispersion in near-shore areas.

The device presented here makes possible to measure the evolution
of the total petroleum hydrocarbon concentration during this period
and our results show no important reduction of TPH concentration
over the 24 hours of contamination (Table 1). Thus, the experimental
approach succeeds in simulating dispersant application in near-shore
areas.

Conclusion

Bioassays must be considered with caution due to the complexity of work with living material [30]. Indeed, numerous settings could
influence the results of the experimentation if they cannot be controlled.
However, during an emergency context such an oil spill, bioassay could
be relevant to evaluate the potential biological impact of operational
response.

Our concern was to find an efficient and reliable method for rapid
toxicity assessment of a dispersed oil spill even in near-shore areas.
This study presents an experimental system designed to perform
reproducible toxicity tests on small marine organisms and to simulate
the increasing transfer of petroleum from the surface to the water
column when dispersant is applied (comparing MD and CD1, CD2).
Moreover comparisons of preliminary toxicity assays performed on
juvenile fish with published literature suggest that the experimental
system is suitable for assessing the toxicity of dispersant application.
Nevertheless, in this system attention must be paid to the fact that a
part of the evolution of TPH concentrations and size of oil droplets are
dependent on the given turbulent mixing energy. This mixing energy
can be changed to respond to different oil spill scenario.

Thus, the present experimental approach seems of interest in order
to establish a comprehensive framework in an emergency context and
especially to dispersant use during an oil spill. However, the natural
environment is complex and full of interaction between biotic and
abiotic parameters. These laboratory results can differ with in situ
observations. Consequently, for a better comprehension of these
interactions, these acute toxicity studies can be coupled with studies
focused on the sub-lethal effects of oil-dispersant mixtures.

Acknowledgements

This study was supported by a PhD grant from the Conseil Général of the
Charente-Maritime. The Agence Nationale de la Recherche is acknowledged for
financial support for the project ‘DISCOBIOL’. The authors also acknowledge Total
Fluides and Innospech for providing chemicals.

AFNOR NF-T (1997). Dispersants, determination of the acute toxicity of a substance -à-vis the marine shrimp (Palaemonetesvarians). French standard published and distributed by the French Association for Standardization (AFNOR) April.